The present invention relates to naphthotriazolium salts, their synthesis and use in the determination of reducing substances.
The present triazolium salts are usually colorless or weakly yellow compounds which upon reduction are converted into azo dyes (R. Kuhn and E. Ludolphy, Liebigs, Ann. Chem. 564, 35-43, (1949). Accordingly, the present compounds are useful for the quantitative determination of reducing substances by photometric methods, e.g., by measuring absorbance or reflectance. A field of application of interest is, for example, the quantitative detection of reducing gases in the atmosphere. Such compounds can be, for example, H.sub.2 S, AsH.sub.3, B.sub.2 H.sub.6 or PH.sub.3. Moreover, other reducing substances can also be detected by reaction with the naphthotriazolium salts of the present invention, for example, organic thiols, ascorbic acid, and biological reducing agents such as NADH or NADPH.
It is well known that reducing pyridine nucleotides react in the presence of N-methyl-phenazinium salts or the enzyme diaphorase with reduction indicators such as, for example, the tetrazolium salts to give colored products (H.U. Bergmeyer, Methods of Enzymatic Analysis, 3rd edition, Vol. I, p. 197 et seq.). A number of assay methods for reduced pyridine nucleotides such as NADH or NADPH are based on these reactions. In the presence of the enzyme diaphorase, NADH reduces a reductive indicator, such as a tetrazolium salt, with the formation of AND to give a dye whose concentration can be determined by a variety of photometric means. By coupling this indicator reaction with various enzymatic redox reactions, a variety of different analytes, for example glucose or cholesterol, can be determined in body fluids.
Triazolium salts, as opposed to the well-known tetrazolium salts, have been suggested for use in the determination of NADH, however, from the literature they are known to have inferior reducibility compared to the tetrazolium salts, and are therefore too slowly reacting [E. Seidler, Acta Histochem. 82, 89-93 (1987)]. Furthermore, in the case of the triazolium salts described in the literature, the absorption maxima of the reduction products are of very short wavelength (.lambda. max&lt;540 nm). Indicators producing dye products compounds having distinctly longer wavelength absorption maxima are desirable in order, for example, to be able to carry out measurements free of hemoglobin or bilirubin interferences.
It is an object of the present invention to provide triazolium salts characterized by easy and rapid reducibility and bathochromic absorption maxima, and which are thus advantageous for use in the chromogenic determination of reducing substances, particularly NADH.